《生物技术与人类》课程教学资源（经典阅读）Assessment of the safety of foods derived from genetically modified

ELSEVIER Food and Chemical Toxicology 42(2004)1047-1088 Po www.elsevier.com/locate/foodchemtox Assessment of the safety of foods derived from genetically modified (GM)crops A.Koniga*,A.Cockburnb,R.W.R.Crevele,E.Debruyned,R.Grafstroeme, U.Hammerlingf,I.Kimber,I.Knudsen,H.A.Kuiper',A.A.C.M.Peijnenburg', A.H.Penninks',M.Poulsen,M.Schauzuk,J.M.Wal Harvard Center for Risk Analysis,Harvard School of Public Health,Harvard University,718 Huntington Avenue,Boston,MA 02115.USA PScientific Affairs,Agricultural Sector,Monsanto Service International.Avenue de Tervuren 270-272,B-1150 Brussels,Belgium SEAC Toxicology Department,Unilever Research Colworth,Colworth House,Sharnbrook,Bedford MK44 ILO,UK Regulatory Toxicology,Herbicides and Biotechnology,Bayer CropScience,355 rue Dostoievski-BP 153.F-06903 Sophia Antipolis Cedex,France Institute of Environmental Medicine,Karolinska Institutet,Nobelsvag 13.Box 210,SE-17177 Stockholm,Sweden Swedish National Food Administration,PO Box 622.SE-751 Uppsala,Sweden ECentral Toxicology Laboratory,Syngenta UK,Alderley Park Macclesfield,Cheshire SK10 4TJ,UK hInstitute of Food Safety and Toxicology.Danish Veterinary and Food Administration.19 Moerkhoej Bygade.DK-2860 Soborg.Denmark RIKILT-Institute of Food Safety,Wageningen University Research Centre,Bornsesteeg 45,PO Box 230, NL-6700 AE Wageningen.The Netherlands iTNO Nutrition and Food Research.Utrechtseweg 48.NL-3700 AJ Zeist.The Netherlands kCentre for Novel Foods and Genetic Engineering,Federal Institute for Risk Assessment,Thielallee 88-92,D-14195 Berlin,Germany Service de Pharmacologie et d'Immunologie,Laboratoire Associe INRA-CEA d'Immuno-Allergie Alimentaire.CEA-SACLAY Batiment 136. F-91191 Gif-sur-Yvette,France Received 15 November 2003;accepted 4 February 2004 Abstract This paper provides guidance on how to assess the safety of foods derived from genetically modified crops(GM crops);it sum- marises conclusions and recommendations of Working Group I of the ENTRANSFOOD project.The paper provides an approach for adapting the test strategy to the characteristics of the modified crop and the introduced trait,and assessing potential unintended effects from the genetic modification.The proposed approach to safety assessment starts with the comparison of the new GM crop with a traditional counterpart that is generally accepted as safe based on a history of human food use (the concept of substantial Abbreviations:ADI,Acceptable Daily Intake;ADME,absorption,distribution,metabolism,and excretion;ALLERGEST,EU-project on the effect of gastrointestinal digestion on the allergenicity of foods;APHIS,Animal and Plant Health Inspection Service;Bt,Bacillus thuringiensis;cDNA, DNA complementary to an RNA strand:CP4 EPSPS,Agrobacterium sp.CP4-derived enolpyruvylshikimate-3-phosphate synthase;DAFNE,Data Food Networking:DNA,deoxyribonucleic acid;EDI,estimated daily intake:EFG,Euro Food Group;EFSA,European Food Safety Authority: ENDB,European Nutrient Database:ENTRANSFOOD,European network safety assessment of genetically modified food crops;EPA,US Environmental Protection Agency:EPIC,European Prospective Investigation into Cancer and Nutrition:EPSPS.enolpyruvylshikimate-3-phos. phate synthase;EU,European Union:FAO,Food and Agriculture Organisation of the United Nations;FDA.US Food and Drug Administration; FOSIE,European Concerted Action Food Safety in Europe;FSANZ,Food Standards Australia New Zealand;GEMS/FOOD,Global Environ- ment Monitoring System/Food Contamination Monitoring and Assessment Programme;GM,genetically modified:GMO,genetically modified organism;IFBC,International Food Biotechnology Council;ILSI,International Life Sciences Institute;INFORMALL,EU-project about com- municating about food allergies and information for consumers,regulators,and industry;INVITTOX,databank of in vitro techniques in toxicol- ogy;MAFF,Japan Ministry of Agriculture,Food,and Fisheries;MHLW,Ministry of Health,Labour,and Welfare;MOS,margin of safety; NOAEL.No Observed Adverse Effect Level:ODE,US FDA Office of Device Evaluation;OECD,Organisation for Economic Co-operation and Development:PCR.polymerase chain reaction:Protall,EU-project on food allergens of plant origin and the relationship between allergenic potential and biological activity:RDA,recommended daily allowance:RNA,ribonucleic acid:SAFOTEST.EU-project on new methods for the safety testing of transgenic food:SGF,simulated gastric fluid:TMDI,theoretical maximum daily intake:UK.United Kingdom;UK ACRE,UK Advisory Committee on Releases into the Environment:US,United States;USA,United States of America;USDA,US Department of Agriculture; WHO.World Health Organisation of the United Nations Corresponding author.Tel.:+1-617-4324497;fax:+1-617-4320190. E-mail address:ariane_koenig@harvard.edu (A.Konig) 0278-6915/S-see front matter C 2004 Elsevier Ltd.All rights reserved. doi:10.1016j.fct.2004.02.019

Assessment of the safety of foods derived from genetically modified (GM) crops A. Ko¨niga,*, A. Cockburnb, R.W.R. Crevelc , E. Debruyned, R. Grafstroeme , U. Hammerlingf , I. Kimberg , I. Knudsenh, H.A. Kuiperi , A.A.C.M. Peijnenburgi , A.H. Penninksj , M. Poulsenh, M. Schauzuk, J.M. Wall a Harvard Center for Risk Analysis, Harvard School of Public Health, Harvard University, 718 Huntington Avenue, Boston, MA 02115, USA bScientific Affairs, Agricultural Sector, Monsanto Service International, Avenue de Tervuren 270-272, B-1150 Brussels, Belgium c SEAC Toxicology Department, Unilever Research Colworth, Colworth House, Sharnbrook, Bedford MK44 1LQ, UK dRegulatory Toxicology, Herbicides and Biotechnology, Bayer CropScience, 355 rue Dostoievski-BP 153, F-06903 Sophia Antipolis Cedex, France e Institute of Environmental Medicine, Karolinska Institutet, Nobelsva¨g 13, Box 210, SE-17177 Stockholm, Sweden f Swedish National Food Administration, PO Box 622, SE-751 Uppsala, Sweden g Central Toxicology Laboratory, Syngenta UK, Alderley Park Macclesfield, Cheshire SK10 4TJ, UK hInstitute of Food Safety and Toxicology, Danish Veterinary and Food Administration, 19 Moerkhoej Bygade, DK-2860 Søborg, Denmark i RIKILT- Institute of Food Safety, Wageningen University & Research Centre, Bornsesteeg 45, PO Box 230, NL-6700 AE Wageningen, The Netherlands j TNO Nutrition and Food Research, Utrechtseweg 48, NL-3700 AJ Zeist, The Netherlands kCentre for Novel Foods and Genetic Engineering, Federal Institute for Risk Assessment, Thielallee 88-92, D-14195 Berlin, Germany l Service de Pharmacologie et d’Immunologie, Laboratoire Associe´ INRA-CEA d’Immuno-Allergie Alimentaire, CEA-SACLAY Baˆtiment 136, F-91191 Gif-sur-Yvette, France Received 15 November 2003; accepted 4 February 2004 Abstract This paper provides guidance on how to assess the safety of foods derived from genetically modified crops (GM crops); it sum￾marises conclusions and recommendations of Working Group 1 of the ENTRANSFOOD project. The paper provides an approach for adapting the test strategy to the characteristics of the modified crop and the introduced trait, and assessing potential unintended effects from the genetic modification. The proposed approach to safety assessment starts with the comparison of the new GM crop with a traditional counterpart that is generally accepted as safe based on a history of human food use (the concept of substantial 0278-6915/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2004.02.019 Food and Chemical Toxicology 42 (2004) 1047–1088 www.elsevier.com/locate/foodchemtox Abbreviations: ADI, Acceptable Daily Intake; ADME, absorption, distribution, metabolism, and excretion; ALLERGEST, EU-project on the effect of gastrointestinal digestion on the allergenicity of foods; APHIS, Animal and Plant Health Inspection Service; Bt, Bacillus thuringiensis; cDNA, DNA complementary to an RNA strand; CP4 EPSPS, Agrobacterium sp. CP4-derived enolpyruvylshikimate-3-phosphate synthase; DAFNE, Data Food Networking; DNA, deoxyribonucleic acid; EDI, estimated daily intake; EFG, Euro Food Group; EFSA, European Food Safety Authority; ENDB, European Nutrient Database; ENTRANSFOOD, European network safety assessment of genetically modified food crops; EPA, US Environmental Protection Agency; EPIC, European Prospective Investigation into Cancer and Nutrition; EPSPS, enolpyruvylshikimate-3-phos￾phate synthase; EU, European Union; FAO, Food and Agriculture Organisation of the United Nations; FDA, US Food and Drug Administration; FOSIE, European Concerted Action Food Safety in Europe; FSANZ, Food Standards Australia New Zealand; GEMS/FOOD, Global Environ￾ment Monitoring System/Food Contamination Monitoring and Assessment Programme; GM, genetically modified; GMO, genetically modified organism; IFBC, International Food Biotechnology Council; ILSI, International Life Sciences Institute; INFORMALL, EU-project about com￾municating about food allergies and information for consumers, regulators, and industry; INVITTOX, databank of in vitro techniques in toxicol￾ogy; MAFF, Japan Ministry of Agriculture, Food, and Fisheries; MHLW, Ministry of Health, Labour, and Welfare; MOS, margin of safety; NOAEL, No Observed Adverse Effect Level; ODE, US FDA Office of Device Evaluation; OECD, Organisation for Economic Co-operation and Development; PCR, polymerase chain reaction; Protall, EU-project on food allergens of plant origin and the relationship between allergenic potential and biological activity; RDA, recommended daily allowance; RNA, ribonucleic acid; SAFOTEST, EU-project on new methods for the safety testing of transgenic food; SGF, simulated gastric fluid; TMDI, theoretical maximum daily intake; UK, United Kingdom; UK ACRE, UK Advisory Committee on Releases into the Environment; US, United States; USA, United States of America; USDA, US Department of Agriculture; WHO, World Health Organisation of the United Nations. * Corresponding author. Tel.: +1-617-4324497; fax: +1-617-4320190. E-mail address: ariane_koenig@harvard.edu (A. Ko¨nig)

1048 A.Konig et al.Food and Chemical Toxicology 42 (2004)1047-1088 equivalence).This case-focused approach ensures that foods derived from GM crops that have passed this extensive test-regime are as safe and nutritious as currently consumed plant-derived foods.The approach is suitable for current and future GM crops with more complex modifications.First,the paper reviews test methods developed for the risk assessment of chemicals,including food additives and pesticides,discussing which of these methods are suitable for the assessment of recombinant proteins and whole foods.Second,the paper presents a systematic approach to combine test methods for the safety assessment of foods derived from a specific GM crop.Third,the paper provides an overview on developments in this area that may prove of use in the safety assess- ment of GM crops,and recommendations for research priorities.It is concluded that the combination of existing test methods provides a sound test-regime to assess the safety of GM crops.Advances in our understanding of molecular biology,biochemistry, and nutrition may in future allow further improvement of test methods that will over time render the safety assessment of foods even more effective and informative. C 2004 Elsevier Ltd.All rights reserved. Keywords:Food;Plant biotechnology;Genetic modification;Genetic engineering:Genetic manipulation;Transgenic crops;Novel foods;Recom- binant proteins;Plant metabolism;Regulation;Safety assessment;Risk analysis;Molecular characterisation;Toxicology;Allergy;Substantial equivalence;Unintended effects;Bioinformatics;In vitro test methods;In vivo test methods;Animal testing;Post market monitoring;Estimated consumption;Exposure assessment:Compositional analysis;Advanced analytical methods;Profiling 1.Introduction complex modifications.The remainder of this paper is divided into four sections.Section 2 provides an over- Approaches to the regulation and safety assessment of view of regulations and internationally agreed principles genetically modified(GM)crops have been developed in and guidelines for risk assessment of chemicals and a very proactive manner.The first international and foods derived from GM crops.Section 3 reviews exist- national provisions for the safety assessment and reg- ing test methods developed for chemicals and food ulation of genetically modified organisms (GMOs), additives,and examines their suitability for testing the including GM crops and derived foods were drawn up safety of foods and food constituents derived from GM by scientific experts in the mid-1980s (OECD,1986;US crops.Section 4 systematically sets out how to deter- OSTP,1986).This was nearly a decade before the first mine whether the GM crop is 'as safe as'a suitable regulatory approval of a genetically modified crop in comparator with a history of human consumption.It 1995.Since then,the global area of commercial cultiva- provides guidance on how to compile information on tion of such crops has risen to 58.7 million hectares in the parent crop and on the genetic modification.This 2002 (James,2002).Commercially cultivated GM crops information in turn guides the choice of test para- include soybean.maize,cotton.canola.potatoes,and meters and methods in the analysis of any introduced tomatoes.At present,the most widely grown GM crops substance and of the whole GM crop.Any significant contain new genes that confer herbicide tolerance or differences that are identified in this systematic compar- insect resistance.Other crops are being developed that ison of the GM crop and the comparator then are sub- have improved nutritional characteristics for their food ject to further investigation as to whether this difference or feed use;GM soybeans and oil seed rape with altered might have implications for human health.Section 5 fatty acid profiles,for example,have already undergone discusses implications of advances in molecular biology regulatory review.Future advances in genomic sciences and the development of in vitro and in vivo test methods promise the discovery of new genes conferring desirable for the future refinement of food safety assessment characteristics to crops that may fundamentally alter a strategies. crop's metabolic functions,promising further nutri- The paper provides detailed guidance for anyone tional enhancement and resistance to abiotic stresses.It involved in risk assessment and regulation of GM crops. is important that we should continue to proactively The paper emphasises that this systematic approach to assess whether current approaches to safety assessment food safety assessment of GM crops offers a high level are appropriate also for future GM crop products with of safety assurance:this iterative and case-focused more complex traits. design of safety testing strategies ensures that all tested This paper presents a systematic approach for com- and approved foods derived from GM crops are as safe bining different test methods to assess the safety of and nutritious as currently consumed plant-derived foods derived from a specific GM crop.It provides foods.The paper also considers how our continuously guidance on how to tailor the test strategy to the improving understanding of molecular biology,bio- characteristics of the modified crop and the introduced chemistry,and nutrition will over time facilitate the trait and identifying potential unintended effects from development of new crop varieties and their safety the genetic modification.The approach builds on assessment.The conclusion provides recommendations internationally agreed guidelines and principles,and is on priorities for research and development of test suitable for current and future GM crops with more methods and strategies

equivalence). This case-focused approach ensures that foods derived from GM crops that have passed this extensive test-regime are as safe and nutritious as currently consumed plant-derived foods. The approach is suitable for current and future GM crops with more complex modifications. First, the paper reviews test methods developed for the risk assessment of chemicals, including food additives and pesticides, discussing which of these methods are suitable for the assessment of recombinant proteins and whole foods. Second, the paper presents a systematic approach to combine test methods for the safety assessment of foods derived from a specific GM crop. Third, the paper provides an overview on developments in this area that may prove of use in the safety assess￾ment of GM crops, and recommendations for research priorities. It is concluded that the combination of existing test methods provides a sound test-regime to assess the safety of GM crops. Advances in our understanding of molecular biology, biochemistry, and nutrition may in future allow further improvement of test methods that will over time render the safety assessment of foods even more effective and informative. # 2004 Elsevier Ltd. All rights reserved. Keywords: Food; Plant biotechnology; Genetic modification; Genetic engineering; Genetic manipulation; Transgenic crops; Novel foods; Recom￾binant proteins; Plant metabolism; Regulation; Safety assessment; Risk analysis; Molecular characterisation; Toxicology; Allergy; Substantial equivalence; Unintended effects; Bioinformatics; In vitro test methods; In vivo test methods; Animal testing; Post market monitoring; Estimated consumption; Exposure assessment; Compositional analysis; Advanced analytical methods; Profiling 1. Introduction Approaches to the regulation and safety assessment of genetically modified (GM) crops have been developed in a very proactive manner. The first international and national provisions for the safety assessment and reg￾ulation of genetically modified organisms (GMOs), including GM crops and derived foods were drawn up by scientific experts in the mid-1980s (OECD, 1986; US OSTP, 1986). This was nearly a decade before the first regulatory approval of a genetically modified crop in 1995. Since then, the global area of commercial cultiva￾tion of such crops has risen to 58.7 million hectares in 2002 (James, 2002). Commercially cultivated GM crops include soybean, maize, cotton, canola, potatoes, and tomatoes. At present, the most widely grown GM crops contain new genes that confer herbicide tolerance or insect resistance. Other crops are being developed that have improved nutritional characteristics for their food or feed use; GM soybeans and oil seed rape with altered fatty acid profiles, for example, have already undergone regulatory review. Future advances in genomic sciences promise the discovery of new genes conferring desirable characteristics to crops that may fundamentally alter a crop’s metabolic functions, promising further nutri￾tional enhancement and resistance to abiotic stresses. It is important that we should continue to proactively assess whether current approaches to safety assessment are appropriate also for future GM crop products with more complex traits. This paper presents a systematic approach for com￾bining different test methods to assess the safety of foods derived from a specific GM crop. It provides guidance on how to tailor the test strategy to the characteristics of the modified crop and the introduced trait and identifying potential unintended effects from the genetic modification. The approach builds on internationally agreed guidelines and principles, and is suitable for current and future GM crops with more complex modifications. The remainder of this paper is divided into four sections. Section 2 provides an over￾view of regulations and internationally agreed principles and guidelines for risk assessment of chemicals and foods derived from GM crops. Section 3 reviews exist￾ing test methods developed for chemicals and food additives, and examines their suitability for testing the safety of foods and food constituents derived from GM crops. Section 4 systematically sets out how to deter￾mine whether the GM crop is ‘as safe as’ a suitable comparator with a history of human consumption. It provides guidance on how to compile information on the parent crop and on the genetic modification. This information in turn guides the choice of test para￾meters and methods in the analysis of any introduced substance and of the whole GM crop. Any significant differences that are identified in this systematic compar￾ison of the GM crop and the comparator then are sub￾ject to further investigation as to whether this difference might have implications for human health. Section 5 discusses implications of advances in molecular biology and the development of in vitro and in vivo test methods for the future refinement of food safety assessment strategies. The paper provides detailed guidance for anyone involved in risk assessment and regulation of GM crops. The paper emphasises that this systematic approach to food safety assessment of GM crops offers a high level of safety assurance: this iterative and case-focused design of safety testing strategies ensures that all tested and approved foods derived from GM crops are as safe and nutritious as currently consumed plant-derived foods. The paper also considers how our continuously improving understanding of molecular biology, bio￾chemistry, and nutrition will over time facilitate the development of new crop varieties and their safety assessment. The conclusion provides recommendations on priorities for research and development of test methods and strategies. 1048 A. Ko¨nig et al. / Food and Chemical Toxicology 42 (2004) 1047–1088

A.Konig et al.Food and Chemical Toxicology 42 (2004)1047-1088 1049 2.Food safety of GM crops:regulation,principles,and into force on 17 October 2002,introduces mandatory guidelines labelling and traceability requirements.It also limits approvals to a period of 10 years;furthermore,appli- 2.1.Regulatory frameworks for GM crops and derived cants have to provide post-market monitoring plans for foods some categories of products. Since 1997 a separate approval procedure for foods Food safety systems,comprising institutions,policies, derived from genetically modiifed organisms exists. laws,and guidelines for assessments,continually evolve Regulation EC No 258/97 concerning novel foods and over time.The evolution of food safety systems in indi- food ingredients in 1997 (hence forth Novel Foods vidual jurisdictions is affected both by science and Regulation)(European Commission,1997a)covers all society:Scientific advances improve our understanding foods that have not hitherto been used for human con- of health implications of foods and lead to adoption of sumption to a significant degree within the European new agri-food production technologies,some of which Community.The European Commission has published require regulatory oversight.Changing societal values guidelines for data and information to be included in can lead to shifts in emphasis in consumer protection applications by petitioners (European Commission, policies and regulatory and institutional change.Reg- 1997b).The Novel Foods Regulation requires the risk ulation in turn can affect both innovation and risk per- assessment and pre-market approval of novel foods, ception.The Organisation for Economic Co-operation and also specifies labelling requirements for certain and Development (OECD)recently compiled descrip- categories of novel foods.The Novel Foods Regulation tions of national food safety systems of its twenty-nine gave the European Commission a clear role in the gov- Member States;these descriptions also specifically ernance of food safety in the European Union. address national approaches to the regulation and This role was strengthened with the publication of the assessment of foods derived from GM crops(OECD, Regulation EC No 178/2002 on the general principles of 2000.2003). food law and the establishment of the European Food Two types of regulatory frameworks for foods derived Safety Authority (hence forth the General Food Law) from GM crops can be distinguished.Some jurisdictions (European Commission,2002;see also European Com- enacted specific 'process-based'legislation for the reg- mission,2000a).The General Food Law provides the ulation of all genetically modified organisms,these legal basis for the establishment of a fully integrated include the European Union (EU)and Australia.In system for food safety legislation and controls covering contrast,other regulatory systems are 'product-based', all aspects of food production and the establishment of focusing on the resulting product characteristics and an independent European Food Safety Authority use,and not on the process of genetic modification,as (EFSA).The General Food Law provides an integrated for instance those in the United States of America approach to ensuring food safety across the EU Mem- (USA)and Canada. ber States and across the food and feed sectors.General In the European Union(EU)the regulation of foods principles of EU food law state that risk analysis is derived from genetically modified organisms has under- based on scientific risk assessment conducted by the gone two significant changes since it first was instituted recently instituted European Food Safety Authority and in the early 1990s.These changes occurred in tandem establish an EU-level authorisation procedure.Other with more fundamental changes in the governance of general principles include the protection and informing food safety in the EU.First,a horizontal,process-based of consumers through comprehensive labelling schemes; law regulating all genetically modified organisms to be provisions for traceability-that is the ability to trace released into the environment came into force in 1990. back to the origin and to understand the distribution of Directive 90/220/EEC governed experimental releases foods and food ingredients;and the application of the and marketing authorisation of all genetically modified precautionary principle in instances of significant organisms(European Commission,1990).The Directive uncertainty in the risk assessment.Furthermore,the set out an approval process requiring the case-by-case new law clarifies accountability of all legal entities assessment of the potential risks to human and animal involved in food production and regulation in the EU health and the environment of all genetically modified by describing general food safety requirements that are organisms or products consisting of or containing a imposed on both the Member States and business GMO (except for pharmaceuticals,which are regulated operators. separately).Partly in response to the public debate on The General Food Law provides for one decision- GM crops,the Directive was revised to strengthen the making procedure for all products that require EU- existing requirements for risk assessment and the deci- level approvals,such as food additives,pesticide resi- sion-making process (European Commission,2001). dues in food,novel foods,and genetically modified The revised Directive 2001/18/EC on the deliberate organisms.The procedure is as follows:The European release of genetically modified organisms,which entered Commission Directorate for Health and Consumer

2. Food safety of GM crops: regulation, principles, and guidelines 2.1. Regulatory frameworks for GMcrops and derived foods Food safety systems, comprising institutions, policies, laws, and guidelines for assessments, continually evolve over time. The evolution of food safety systems in indi￾vidual jurisdictions is affected both by science and society: Scientific advances improve our understanding of health implications of foods and lead to adoption of new agri-food production technologies, some of which require regulatory oversight. Changing societal values can lead to shifts in emphasis in consumer protection policies and regulatory and institutional change. Reg￾ulation in turn can affect both innovation and risk per￾ception. The Organisation for Economic Co-operation and Development (OECD) recently compiled descrip￾tions of national food safety systems of its twenty-nine Member States; these descriptions also specifically address national approaches to the regulation and assessment of foods derived from GM crops (OECD, 2000, 2003). Two types of regulatory frameworks for foods derived from GM crops can be distinguished. Some jurisdictions enacted specific ‘process-based’ legislation for the reg￾ulation of all genetically modified organisms, these include the European Union (EU) and Australia. In contrast, other regulatory systems are ‘product-based’, focusing on the resulting product characteristics and use, and not on the process of genetic modification, as for instance those in the United States of America (USA) and Canada. In the European Union (EU) the regulation of foods derived from genetically modified organisms has under￾gone two significant changes since it first was instituted in the early 1990s. These changes occurred in tandem with more fundamental changes in the governance of food safety in the EU. First, a horizontal, process-based law regulating all genetically modified organisms to be released into the environment came into force in 1990. Directive 90/220/EEC governed experimental releases and marketing authorisation of all genetically modified organisms (European Commission, 1990). The Directive set out an approval process requiring the case-by-case assessment of the potential risks to human and animal health and the environment of all genetically modified organisms or products consisting of or containing a GMO (except for pharmaceuticals, which are regulated separately). Partly in response to the public debate on GM crops, the Directive was revised to strengthen the existing requirements for risk assessment and the deci￾sion-making process (European Commission, 2001). The revised Directive 2001/18/EC on the deliberate release of genetically modified organisms, which entered into force on 17 October 2002, introduces mandatory labelling and traceability requirements. It also limits approvals to a period of 10 years; furthermore, appli￾cants have to provide post-market monitoring plans for some categories of products. Since 1997 a separate approval procedure for foods derived from genetically modiifed organisms exists. Regulation EC No 258/97 concerning novel foods and food ingredients in 1997 (hence forth Novel Foods Regulation) (European Commission, 1997a) covers all foods that have not hitherto been used for human con￾sumption to a significant degree within the European Community. The European Commission has published guidelines for data and information to be included in applications by petitioners (European Commission, 1997b). The Novel Foods Regulation requires the risk assessment and pre-market approval of novel foods, and also specifies labelling requirements for certain categories of novel foods. The Novel Foods Regulation gave the European Commission a clear role in the gov￾ernance of food safety in the European Union. This role was strengthened with the publication of the Regulation EC No 178/2002 on the general principles of food law and the establishment of the European Food Safety Authority (hence forth the General Food Law) (European Commission, 2002; see also European Com￾mission, 2000a). The General Food Law provides the legal basis for the establishment of a fully integrated system for food safety legislation and controls covering all aspects of food production and the establishment of an independent European Food Safety Authority (EFSA). The General Food Law provides an integrated approach to ensuring food safety across the EU Mem￾ber States and across the food and feed sectors. General principles of EU food law state that risk analysis is based on scientific risk assessment conducted by the recently instituted European Food Safety Authority and establish an EU-level authorisation procedure. Other general principles include the protection and informing of consumers through comprehensive labelling schemes; provisions for traceability-that is the ability to trace back to the origin and to understand the distribution of foods and food ingredients; and the application of the precautionary principle in instances of significant uncertainty in the risk assessment. Furthermore, the new law clarifies accountability of all legal entities involved in food production and regulation in the EU by describing general food safety requirements that are imposed on both the Member States and business operators. The General Food Law provides for one decision￾making procedure for all products that require EU￾level approvals, such as food additives, pesticide resi￾dues in food, novel foods, and genetically modified organisms. The procedure is as follows: The European Commission Directorate for Health and Consumer A. Ko¨nig et al. / Food and Chemical Toxicology 42 (2004) 1047–1088 1049

1050 A.Konig et al.Food and Chemical Toxicology 42 (2004)1047-1088 Protection administers the review process.The Eur- The USDA regulates the import,interstate movement, opean Food Safety Authority reviews the risk assess- field trial release,and commercial release of GM crops ment submitted by applicants intending to place a under the Federal Plant Pest Act and the Plant Novel Food on the European market.It is up to the Quarantine Act,which are administered by the Animal administrators in the European Commission Directo- and Plant Health Inspection Service (APHIS).Prior to rate General for Health and Consumer Protection to approval for unrestricted release,as in commercialisa- draft proposals based on the risk assessment and other tion,the USDA/APHIS must determine that the GM broader considerations that may affect choice of policy crop is not a plant pest.There is no Federal regulation options.A regulatory committee of representatives of requiring the registration of new plant varieties.The Member States competent authorities then decides EPA has regulatory oversight for all GM crops that whether to accept the Commission proposal through a produce a plant pesticide.Plant-integrated pesticides are weighted voting system.If the regulatory committee's regulated according to the same procedures as other opinion is not in accordance with the proposed pesticides. measure or if no opinion is delivered,the question is The FDA has authority over human food and animal referred to the Council of Ministers.The Council of feed safety and the wholesomeness of all plant products, Ministers can approve or reject a Commission proposal including those produced via genetic modification, given a qualified majority of member States support under the Federal Food Drug and Cosmetic Act.The the position.If rejected,the European Commission has FDA has concluded that food and feed derived from to prepare a new proposal.If the Council of Ministers GM crops pose no unique safety concerns and,there- takes no decision within three months,or does not fore,that the food and feed products derived from these reach a qualified majority indicating that it opposes the plants should be regulated no differently than compar- proposal,the European Commission shall adopt the able products derived from traditional plant breeding or proposal. any other genetic modification approach (US FDA, In June 2003 the European Council of Ministers 1992).Labelling is only mandated for foods that present adopted two new Regulations specific for foods and a health risk to subgroups of the population,such as feeds derived from genetically modified organisms. allergenic foods:the FDA does not mandate process- Regulation (EC)No 1829/2003 on genetically modified based labelling informing consumers for instance on a food and feed provides the legal basis for the approval food's content of genetically modified organisms.Partly procedure for genetically modified organisms as speci- in response to demonstrations by activists against GM fied in the General Food Law.The safety of foods crops in Seattle,Washington in 1999,and to three pub- derived from genetically modified organisms is assessed lic hearings,the FDA decided to adopt measures to by the European Food Safety Authority's Scientific strengthen the scientific basis and transparency of its Panel on genetically modified organisms (European decision-making process.The FDA proposed to modify Commission,2003a).[The panel assesses the food its voluntary process so as to establish mandatory pre- safety,environmental and animal health aspects of market notification and to make its decision process genetically modified organisms ('one-door-one-key' more transparent.The agency also developed draft gui- principle.)]Regulation (EC)No 1830/2003 concerning dance for food manufacturers who wish to label their the traceability and labelling of genetically modified foods voluntarily. organisms and the traceability of food and feed pro- Other systems that present variations of either the US ducts produced from genetically modified organisms or the EU approach are Canada,Australia,and Japan. requires traceability and labelling of genetically mod- In Canada all plants with novel traits are regulated, ified organisms and derived products (European Com- regardless of whether a plant with novel traits was pro- mission,2003b);this regulation also provides a legal duced by conventional breeding,mutagenesis,or basis for case-by-case decisions on post market mon- recombinant DNA techniques (Health Canada,1994; itoring requirements where deemed necessary. CFIA,1998).Foods derived from GM crops are con- The US regulatory framework for GM crops was laid sidered novel foods under the Food and Drugs Act out in the 1986 'Coordinated Framework for Regula- (CFIA,1998).The Canadian Biotechnology Advisory tion of Biotechnology'(US OSTP,1986).Existing laws Committee recently reviewed the Canadian regulations for the regulation of plant pests,pesticides and foods of GM foods;its recommendations include that were amended,resulting in a vertical,product-based research be carried out in order to monitor for hypo- regulatory framework for GM crops and derived foods. thetical long-term health effects(CBAC,2002). Three principal regulatory agencies conduct science- In Japan,the Ministry of Agriculture,Food,and based assessments of risks to human health and the Fisheries (MAFF)and the Ministry of Health,Labour, environment:the United States Department of Agri- and Welfare (MHLW)administer the regulation of food culture (USDA),the Environmental Protection Agency safety of GMOs,including GM crops and other foods (EPA),and the Food and Drug Administration(FDA). and food additives that contain organisms or have been

Protection administers the review process. The Eur￾opean Food Safety Authority reviews the risk assess￾ment submitted by applicants intending to place a Novel Food on the European market. It is up to the administrators in the European Commission Directo￾rate General for Health and Consumer Protection to draft proposals based on the risk assessment and other broader considerations that may affect choice of policy options. A regulatory committee of representatives of Member States competent authorities then decides whether to accept the Commission proposal through a weighted voting system. If the regulatory committee’s opinion is not in accordance with the proposed measure or if no opinion is delivered, the question is referred to the Council of Ministers. The Council of Ministers can approve or reject a Commission proposal given a qualified majority of member States support the position. If rejected, the European Commission has to prepare a new proposal. If the Council of Ministers takes no decision within three months, or does not reach a qualified majority indicating that it opposes the proposal, the European Commission shall adopt the proposal. In June 2003 the European Council of Ministers adopted two new Regulations specific for foods and feeds derived from genetically modified organisms. Regulation (EC) No 1829/2003 on genetically modified food and feed provides the legal basis for the approval procedure for genetically modified organisms as speci- fied in the General Food Law. The safety of foods derived from genetically modified organisms is assessed by the European Food Safety Authority’s Scientific Panel on genetically modified organisms (European Commission, 2003a). [The panel assesses the food safety, environmental and animal health aspects of genetically modified organisms (‘one-door-one-key’ principle.)] Regulation (EC) No 1830/2003 concerning the traceability and labelling of genetically modified organisms and the traceability of food and feed pro￾ducts produced from genetically modified organisms requires traceability and labelling of genetically mod￾ified organisms and derived products (European Com￾mission, 2003b); this regulation also provides a legal basis for case-by-case decisions on post market mon￾itoring requirements where deemed necessary. The US regulatory framework for GM crops was laid out in the 1986 ‘Coordinated Framework for Regula￾tion of Biotechnology’ (US OSTP, 1986). Existing laws for the regulation of plant pests, pesticides and foods were amended, resulting in a vertical, product-based regulatory framework for GM crops and derived foods. Three principal regulatory agencies conduct science￾based assessments of risks to human health and the environment: the United States Department of Agri￾culture (USDA), the Environmental Protection Agency (EPA), and the Food and Drug Administration (FDA). The USDA regulates the import, interstate movement, field trial release, and commercial release of GM crops under the Federal Plant Pest Act and the Plant Quarantine Act, which are administered by the Animal and Plant Health Inspection Service (APHIS). Prior to approval for unrestricted release, as in commercialisa￾tion, the USDA/APHIS must determine that the GM crop is not a plant pest. There is no Federal regulation requiring the registration of new plant varieties. The EPA has regulatory oversight for all GM crops that produce a plant pesticide. Plant-integrated pesticides are regulated according to the same procedures as other pesticides. The FDA has authority over human food and animal feed safety and the wholesomeness of all plant products, including those produced via genetic modification, under the Federal Food Drug and Cosmetic Act. The FDA has concluded that food and feed derived from GM crops pose no unique safety concerns and, there￾fore, that the food and feed products derived from these plants should be regulated no differently than compar￾able products derived from traditional plant breeding or any other genetic modification approach (US FDA, 1992). Labelling is only mandated for foods that present a health risk to subgroups of the population, such as allergenic foods; the FDA does not mandate process￾based labelling informing consumers for instance on a food’s content of genetically modified organisms. Partly in response to demonstrations by activists against GM crops in Seattle, Washington in 1999, and to three pub￾lic hearings, the FDA decided to adopt measures to strengthen the scientific basis and transparency of its decision-making process. The FDA proposed to modify its voluntary process so as to establish mandatory pre￾market notification and to make its decision process more transparent. The agency also developed draft gui￾dance for food manufacturers who wish to label their foods voluntarily. Other systems that present variations of either the US or the EU approach are Canada, Australia, and Japan. In Canada all plants with novel traits are regulated, regardless of whether a plant with novel traits was pro￾duced by conventional breeding, mutagenesis, or recombinant DNA techniques (Health Canada, 1994; CFIA, 1998). Foods derived from GM crops are con￾sidered novel foods under the Food and Drugs Act (CFIA, 1998). The Canadian Biotechnology Advisory Committee recently reviewed the Canadian regulations of GM foods; its recommendations include that research be carried out in order to monitor for hypo￾thetical long-term health effects (CBAC, 2002). In Japan, the Ministry of Agriculture, Food, and Fisheries (MAFF) and the Ministry of Health, Labour, and Welfare (MHLW) administer the regulation of food safety of GMOs, including GM crops and other foods and food additives that contain organisms or have been 1050 A. Ko¨nig et al. / Food and Chemical Toxicology 42 (2004) 1047–1088

A.Konig et al.Food and Chemical Toxicology 42 (2004)1047-1088 1051 obtained through recombinant DNA techniques.The risk.Examples of risk management for conditional food safety assessment of genetically modified organ- approvals include labelling requirements to inform the isms is mandatory under the Specifications and Stan- target group at risk,as done for food products that dards for Food and Food Additives and Other Related contain major allergens.Risk communication is defined Products and is conducted according to guidelines pub- as the exchange of information and opinion on risk lished by the Ministry of Health and Welfare (Japan between risk assessors,risk managers,other interested MHLW,2000).The definition of recombinant DNA parties,and the general public (FAO/WHO,1995, pertains to the introduction of foreign DNA from 1997). sources other than the host;"self cloning"is exempt Some critics voice concerns that the separation of risk from the assessment (Japan MAFF,1995). assessment and risk management neglects that risks are In Australia and New Zealand,the Food Standards also a product of societal circumstances:the salience of Australia New Zealand (FSANZ)has regulatory over- expert advice to concerns of policy makers and the sight over food safety,including the safety of foods public is thus potentially reduced (Jasanoff,1990:NRC. derived from genetically modified organisms.Food 1994,1996;Presidential/Congressional Commission on Standard 1.5.2 specifically regulates the marketing of Risk Assessment and Risk Management,1997;James et foods derived using recombinant DNA techniques al,1999). (FSANZ,2000).The standard also provides for the The focus of this paper is on risk assessment,defined possible post-market monitoring requirements for foods as the evaluation of the probability of known or poten- derived from genetically modified organisms on a case- tial adverse health effects arising from human or animal by-case basis,in particular for foods derived from GM exposure to the identified hazards (FAO/WHO,1995, crops the nutritional characteristics of which were 1997).Such evaluation will always be a central part in modified (FSANZ,2001). the regulation of health risks,regardless of who frames Whilst regulatory frameworks differ across jurisdic- the questions and how broad the assessment is.Risk tions,the approaches to the safety assessment of foods assessment involves combining information on severity derived from GM crops are similar in most countries,as of the consequences of exposure to a hazard and expec- they are based on general principles for risk analysis ted degree of exposure.The first stage in risk assessment and international guidelines for the safety assessment of is to identify the hazards posed by a substance,by foods derived from genetically modified organisms establishing a cause-effect relationship between the hazard and the product or process using toxicological 2.2.General principles of risk analysis experiments,modelling and/or epidemiological meth- ods.It establishes the intrinsic potential of a substance, The general principles for risk analysis were first such as a chemical,protein,or food,to cause adverse established for evaluation of health effects from poten- health effects. tially toxic chemicals.Risk is defined as the likelihood Hazard characterisation aims to evaluate in qualita- that,under particular conditions of exposure,an intrin- tive and quantitative terms the nature of the identified sic hazard will represent a threat to human health.Risk intrinsic hazard.This usually involves an analysis of the is thus a function of hazard and exposure.Hazard is dose-response relationship of harmful effects in the tar- defined as the intrinsic potential of a material to cause get organism or an appropriate surrogate species and adverse health effects:implicit in the definition is the characterisation of the severity of the effect.In routine concept of severity and adversity of the effect.This toxicological studies animals are usually administered definition is consistent with internationally accepted three different doses,including very small doses and principles(FAO/WHO,1995;FAO/WHO,1997;Codex doses that exceed anticipated human exposures by sev- Alimentarius Commission,2003a). eral orders of magnitude.The purpose of these studies is The international principles and guidelines,as well as the establishment of the highest dose level at which no most European policy documents on risk analysis and adverse effect occurs-the No Observed Adverse Effect food safety (see for example European Commission, Level (NOAEL).Animal-based toxicological methods 2000b),draw a distinction between science-based risk for hazard identification and characterisation have assessment usually conducted by experts,risk manage- recently been reviewed by Barlow et al.(2002).The ment,and risk communication.Risk management is NOAEL in the most sensitive animal species in which defined as "the process of weighing policy alternatives tests were conducted is the basis for establishing best to mitigate risks in the light of risk assessment and,if estimates of a safe exposure level for humans.This esti- required,selecting and implementing appropriate con- mation takes into account variations in susceptibility trol options,including regulatory measures"(FAO/ between animal species,and between individuals within WHO,1995;FAO/WHO,1997).Risk management the human population.The observed NOAEL is divi- strategies include authorisation,and implementation of ded by uncertainty factors to establish a margin of risk management measures to minimise or prevent the safety:the default uncertainty factors to account for

obtained through recombinant DNA techniques. The food safety assessment of genetically modified organ￾isms is mandatory under the Specifications and Stan￾dards for Food and Food Additives and Other Related Products and is conducted according to guidelines pub￾lished by the Ministry of Health and Welfare (Japan MHLW, 2000). The definition of recombinant DNA pertains to the introduction of foreign DNA from sources other than the host; ‘‘self cloning’’ is exempt from the assessment (Japan MAFF, 1995). In Australia and New Zealand, the Food Standards Australia New Zealand (FSANZ) has regulatory over￾sight over food safety, including the safety of foods derived from genetically modified organisms. Food Standard 1.5.2 specifically regulates the marketing of foods derived using recombinant DNA techniques (FSANZ, 2000). The standard also provides for the possible post-market monitoring requirements for foods derived from genetically modified organisms on a case￾by-case basis, in particular for foods derived from GM crops the nutritional characteristics of which were modified (FSANZ, 2001). Whilst regulatory frameworks differ across jurisdic￾tions, the approaches to the safety assessment of foods derived from GM crops are similar in most countries, as they are based on general principles for risk analysis and international guidelines for the safety assessment of foods derived from genetically modified organisms. 2.2. General principles of risk analysis The general principles for risk analysis were first established for evaluation of health effects from poten￾tially toxic chemicals. Risk is defined as the likelihood that, under particular conditions of exposure, an intrin￾sic hazard will represent a threat to human health. Risk is thus a function of hazard and exposure. Hazard is defined as the intrinsic potential of a material to cause adverse health effects; implicit in the definition is the concept of severity and adversity of the effect. This definition is consistent with internationally accepted principles (FAO/WHO, 1995; FAO/WHO, 1997; Codex Alimentarius Commission, 2003a). The international principles and guidelines, as well as most European policy documents on risk analysis and food safety (see for example European Commission, 2000b), draw a distinction between science-based risk assessment usually conducted by experts, risk manage￾ment, and risk communication. Risk management is defined as ‘‘the process of weighing policy alternatives to mitigate risks in the light of risk assessment and, if required, selecting and implementing appropriate con￾trol options, including regulatory measures’’ (FAO/ WHO, 1995; FAO/WHO, 1997). Risk management strategies include authorisation, and implementation of risk management measures to minimise or prevent the risk. Examples of risk management for conditional approvals include labelling requirements to inform the target group at risk, as done for food products that contain major allergens. Risk communication is defined as the exchange of information and opinion on risk between risk assessors, risk managers, other interested parties, and the general public (FAO/WHO, 1995, 1997). Some critics voice concerns that the separation of risk assessment and risk management neglects that risks are also a product of societal circumstances; the salience of expert advice to concerns of policy makers and the public is thus potentially reduced (Jasanoff, 1990; NRC, 1994, 1996; Presidential/Congressional Commission on Risk Assessment and Risk Management, 1997; James et al., 1999). The focus of this paper is on risk assessment, defined as the evaluation of the probability of known or poten￾tial adverse health effects arising from human or animal exposure to the identified hazards (FAO/WHO, 1995, 1997). Such evaluation will always be a central part in the regulation of health risks, regardless of who frames the questions and how broad the assessment is. Risk assessment involves combining information on severity of the consequences of exposure to a hazard and expec￾ted degree of exposure. The first stage in risk assessment is to identify the hazards posed by a substance, by establishing a cause-effect relationship between the hazard and the product or process using toxicological experiments, modelling and/or epidemiological meth￾ods. It establishes the intrinsic potential of a substance, such as a chemical, protein, or food, to cause adverse health effects. Hazard characterisation aims to evaluate in qualita￾tive and quantitative terms the nature of the identified intrinsic hazard. This usually involves an analysis of the dose-response relationship of harmful effects in the tar￾get organism or an appropriate surrogate species and characterisation of the severity of the effect. In routine toxicological studies animals are usually administered three different doses, including very small doses and doses that exceed anticipated human exposures by sev￾eral orders of magnitude. The purpose of these studies is the establishment of the highest dose level at which no adverse effect occurs—the No Observed Adverse Effect Level (NOAEL). Animal-based toxicological methods for hazard identification and characterisation have recently been reviewed by Barlow et al. (2002). The NOAEL in the most sensitive animal species in which tests were conducted is the basis for establishing best estimates of a safe exposure level for humans. This esti￾mation takes into account variations in susceptibility between animal species, and between individuals within the human population. The observed NOAEL is divi￾ded by uncertainty factors to establish a margin of safety; the default uncertainty factors to account for A. Ko¨nig et al. / Food and Chemical Toxicology 42 (2004) 1047–1088 1051

1052 A.Konig et al.Food and Chemical Toxicology 42 (2004)1047-1088 variation between species and individuals are both ten. structurally the same and as the transfer of genetic The NOAEL is therefore often divided by 100 to estab- material across species barriers occurs not only in lish the margin of safety.This information then con- laboratories,but also has been a major driving force in stitutes the basis for a determination of a reference dose, evolution.Hence,concepts were developed to focus exposure to which is deemed safe.The amount of a the safety assessment of GM crops on any functional substance exposure to which over a life time is deemed and chemical changes that result from the genetic safe is the Acceptable Daily Intake (ADI).Risk assess- modification. ment requires judgements on what data are considered Foods prepared and used in traditional ways,and sufficient and what uncertainties need to be taken into consumed under anticipated conditions are generally account. regarded as safe based on their history of human con- Information on the quantity and distribution of a sumption,even though they may contain natural toxins potentially hazardous substance in the environment is or anti-nutritional substances,such as neurotoxic then required in order to determine where populations glycoalkaloids in potatoes,or carcinogenic coumarins are expected to come into contact with the substance; in courgettes.The assessment of novel foods,including for foods,dietary intake assessments of populations are foods derived from a GM crop,relies on the use of a required for exposure estimates.Particular attention is food generally recognised as safe as a comparator paid to expected average and worst-case exposure levels (FAO/WHO,1991).The term 'assessment of substantial of the most sensitive subgroups of a population.This equivalence'describes this comparative assessment information is used to determine the population groups approach.This term was first coined by the Office of that may be at risk and the distribution of such risks. Device Evaluation (ODE)of US FDA in the context of These are the elements that allow estimation of the the evaluation of new medical devices that have a com- probability that harm will occur.Exposure assessment parable function to existing medical devices (Miller, often needs to take into account important societal fac- 1999).Authorities and agencies involved in food safety tors necessary to anticipate behaviour of a wide range of assessment in most countries have based their safety individuals that might affect their exposure. assessment strategies and guidelines on this approach Risk characterisation then combines information on (UK Department of Health,1991;US FDA,1992; hazard and exposure.This includes the probable extent, Health Canada,1994;Japan MHLW,2000;European nature,and duration of exposure with considerations of Commission 1997b). hazard characteristics and relevance of those hazards Application of the concept of substantial equivalence for humans in order to estimate the likely risk to human requires the comparison of the GM crop and an appro- health.Any uncertainties inherent in the risk assessment priate 'safe'comparator according to the agronomical should be highlighted.If the expected exposure exceeds and morphological characteristics.and the chemical the established reference dose that was deemed to be composition,including macro-and micro-nutrients,key safe,this has implications for risk management deci- toxins,and key anti-nutrients.This allows identification sions:risk mitigation measures for chemicals can of significant differences between the GM crop and the include measures such as prescribing use of personal comparator,usually the traditionally-bred parent crop protective equipment or restrictions on conditions of (OECD,1993a).Compositional parameters are then use of the chemical:risk management measures for selected that are typical for the crop that is assessed and foods can include labelling,as is the case for allergenic representative of the main metabolic pathways.Sig- foods.The general principles of risk analysis as descri- nificant changes in these parameters are expected to be bed above apply to the safety assessment of foods indicative of any more fundamental changes in the crop derived from GM crops. that need to be evaluated for their potential to have adverse consequence to human health. 2.3.The concept of safety assessment of foods derived The hazard identification and characterisation of GM from GM crops crops therefore is conducted in four steps:(i)Char- acterisation of the parent crop and any hazards asso- The techniques of molecular biology allow the trans- ciated with it:(ii)characterisation of the transformation fer of genes from one organism to another without process and of inserted recombinant DNA (the poten- sexual reproduction and across species.This process tial consequences of any gene transfer event of the allows desirable alterations to be introduced into plant recombinant DNA to microbes or humans should also genomes in a more specific and controlled manner than be assessed);(iii)characterisation of the introduced can be achieved through conventional breeding and proteins (their potential toxicity and allergenicity)and selection of crops.International panels of experts metabolites;and(iv)identification of any other targeted deemed that there are no risks inherent in the use of and unexpected alterations in the GM crop,including recombinant DNA technologies (OECD,1986;Royal changes in the plant metabolism resulting in composi- Society,1998,2002),as all DNA is chemically and tional changes and assessment of their toxicological

variation between species and individuals are both ten. The NOAEL is therefore often divided by 100 to estab￾lish the margin of safety. This information then con￾stitutes the basis for a determination of a reference dose, exposure to which is deemed safe. The amount of a substance exposure to which over a life time is deemed safe is the Acceptable Daily Intake (ADI). Risk assess￾ment requires judgements on what data are considered sufficient and what uncertainties need to be taken into account. Information on the quantity and distribution of a potentially hazardous substance in the environment is then required in order to determine where populations are expected to come into contact with the substance; for foods, dietary intake assessments of populations are required for exposure estimates. Particular attention is paid to expected average and worst-case exposure levels of the most sensitive subgroups of a population. This information is used to determine the population groups that may be at risk and the distribution of such risks. These are the elements that allow estimation of the probability that harm will occur. Exposure assessment often needs to take into account important societal fac￾tors necessary to anticipate behaviour of a wide range of individuals that might affect their exposure. Risk characterisation then combines information on hazard and exposure. This includes the probable extent, nature, and duration of exposure with considerations of hazard characteristics and relevance of those hazards for humans in order to estimate the likely risk to human health. Any uncertainties inherent in the risk assessment should be highlighted. If the expected exposure exceeds the established reference dose that was deemed to be safe, this has implications for risk management deci￾sions: risk mitigation measures for chemicals can include measures such as prescribing use of personal protective equipment or restrictions on conditions of use of the chemical; risk management measures for foods can include labelling, as is the case for allergenic foods. The general principles of risk analysis as descri￾bed above apply to the safety assessment of foods derived from GM crops. 2.3. The concept of safety assessment of foods derived from GMcrops The techniques of molecular biology allow the trans￾fer of genes from one organism to another without sexual reproduction and across species. This process allows desirable alterations to be introduced into plant genomes in a more specific and controlled manner than can be achieved through conventional breeding and selection of crops. International panels of experts deemed that there are no risks inherent in the use of recombinant DNA technologies (OECD, 1986; Royal Society, 1998, 2002), as all DNA is chemically and structurally the same and as the transfer of genetic material across species barriers occurs not only in laboratories, but also has been a major driving force in evolution. Hence, concepts were developed to focus the safety assessment of GM crops on any functional and chemical changes that result from the genetic modification. Foods prepared and used in traditional ways, and consumed under anticipated conditions are generally regarded as safe based on their history of human con￾sumption, even though they may contain natural toxins or anti-nutritional substances, such as neurotoxic glycoalkaloids in potatoes, or carcinogenic coumarins in courgettes. The assessment of novel foods, including foods derived from a GM crop, relies on the use of a food generally recognised as safe as a comparator (FAO/WHO, 1991). The term ‘assessment of substantial equivalence’ describes this comparative assessment approach. This term was first coined by the Office of Device Evaluation (ODE) of US FDA in the context of the evaluation of new medical devices that have a com￾parable function to existing medical devices (Miller, 1999). Authorities and agencies involved in food safety assessment in most countries have based their safety assessment strategies and guidelines on this approach (UK Department of Health, 1991; US FDA, 1992; Health Canada, 1994; Japan MHLW, 2000; European Commission 1997b). Application of the concept of substantial equivalence requires the comparison of the GM crop and an appro￾priate ‘safe’ comparator according to the agronomical and morphological characteristics, and the chemical composition, including macro- and micro-nutrients, key toxins, and key anti-nutrients. This allows identification of significant differences between the GM crop and the comparator, usually the traditionally-bred parent crop (OECD, 1993a). Compositional parameters are then selected that are typical for the crop that is assessed and representative of the main metabolic pathways. Sig￾nificant changes in these parameters are expected to be indicative of any more fundamental changes in the crop that need to be evaluated for their potential to have adverse consequence to human health. The hazard identification and characterisation of GM crops therefore is conducted in four steps: (i) Char￾acterisation of the parent crop and any hazards asso￾ciated with it; (ii) characterisation of the transformation process and of inserted recombinant DNA (the poten￾tial consequences of any gene transfer event of the recombinant DNA to microbes or humans should also be assessed); (iii) characterisation of the introduced proteins (their potential toxicity and allergenicity) and metabolites; and (iv) identification of any other targeted and unexpected alterations in the GM crop, including changes in the plant metabolism resulting in composi￾tional changes and assessment of their toxicological, 1052 A. Ko¨nig et al. / Food and Chemical Toxicology 42 (2004) 1047–1088

A.Konig et al.Food and Chemical Toxicology 42 (2004)1047-1088 1053 allergenic,or nutritional impact.The exposure assess- represents the best available assessment paradigm;no ment includes estimating the dietary intake of the alternative approaches for the safety assessment of new food derived from GM crops and anticipating foods derived from GM crops have been proposed the effect of food processing on any of the introduced (FAO/WHO,2000;Codex Alimentarius Commission, changes. 2003b).Detailed international guidelines for choice of The successful application of the concept of sub- comparators and for best practices in statistical analysis stantial equivalence largely depends on three critical have been and are being established under the auspices elements:the availability of an appropriate comparator of the Organisation for Economic Coordination and and an understanding of the range of variation to be Development (see also Section 4.1). expected within the measured characteristics of that In summary,the concept of substantial equivalence is comparator;the choice of parameters in the single con- widely accepted by international and national agencies stituent compound analyses,the number and type of as the best available guidance for the safety assessment which will strongly influence the validity of any conclu- of new GM crops.The approach recognises that foods sions on comparative safety;and the ability to dis- are complex matrices containing tens of thousands of criminate between differences in the GM crop and the individual constituents,and that their safety assessment comparator that result from the genetic modification therefore requires a comparative approach focusing on and those differences in the plant's germplasm,some of those parameters deemed indicative of the normal which may be attributed to soma-clonal variation functioning of the plant and its metabolism (including introduced during tissue culture,and environmental or biosynthesis of any compound that might affect human cultivation conditions.All such changes that might have health).As with all scientific concepts,the concept of health implications warrant further investigation,even substantial equivalence is evolving and,together with if they can not only be attributed to the genetic modifi- guidelines,making its application more systematic.The cation,unless they are not manifested in subsequent assessment helps to determine whether the GM crop is generations foreseen for commercial cultivation. 'as safe as'its conventional counterpart.Dialogue Any identified differences are then further assessed as between experts and civil society will contribute over to whether they might have adverse implications for time to further clarify and structure risk analysis strate- human health in the range of exposure scenarios.The gies to improve the salience of assessments to address concept of substantial equivalence is thus the starting concerns of policy makers and the public (Tait,2001; point and guiding concept for the safety assessment,not Jasanoff,2000;Schauzu,2000). its conclusion (FAO/WHO.2000:Codex Alimentarius Commission.2003b).If there are no significant differ- ences between the GM crop and the comparator or if 3.Methods for toxicity testing there are differences that will,with reasonable certainty, not adversely affect health,the GM product is con- Regulatory requirements for chemicals such as food sidered 'as safe as'its counterpart.This approach also additives and pesticides,many of which were first insti- applies to GM crops with more complex metabolic tuted in the 1970s.have led to the development of a modifications,where no single parent crop might be a battery of tests to assess the safety of chemicals in foods. suitable comparator,but where single widely consumed Strategies for assessing the food safety of chemicals substances,food constituents.ingredients.or other often combine three approaches:investigation of the whole foods that are deemed safe under representative structure/function relationship for indications of poten- conditions of use may serve as comparators. tial toxicity and allergenicity;in vitro assays with Critics of the concept of substantial equivalence claim enzymes,receptor proteins,or cultured cell lines:and in that current testing approaches do not sufficiently vivo animal studies.The selection of animal studies is address putative unintended and unexpected effects and based on considerations including a molecule's struc- can not rule out the occurrence of potential long-term ture,function and in vitro toxicity results,as well as effects that result from sustained human exposure to ethical criteria.Of these three distinct approaches,evi- such crops that might have subtle compositional chan- dence from animal tests is usually most indicative of ges that may be difficult to detect (Millstone et al., potential toxic effects of a test substance in humans. 1999).Furthermore,some critics maintain that there is a However,the extrapolation of results from animal tests lack of detailed international standards guiding the choice to humans is uncertain.unpredictable differences can of parameters to be measured in the comparative ana- include inter-species and inter-individual differences in lysis and in the application of rigorous statistical analy- metabolism,physiological processes,and lifestyle.These sis,reducing the quality of individual assessments(SBC, uncertainties are usually addressed through the use of 2001).Groups of international experts have reviewed uncertainty factors (see Section 2.2).Individual tox- the concept of substantial equivalence in the light of icological tests can be designed to be specific to test an these criticisms.It was concluded that the concept still hypothesis of a molecule's toxic effect on one particular

allergenic, or nutritional impact. The exposure assess￾ment includes estimating the dietary intake of the new food derived from GM crops and anticipating the effect of food processing on any of the introduced changes. The successful application of the concept of sub￾stantial equivalence largely depends on three critical elements: the availability of an appropriate comparator and an understanding of the range of variation to be expected within the measured characteristics of that comparator; the choice of parameters in the single con￾stituent compound analyses, the number and type of which will strongly influence the validity of any conclu￾sions on comparative safety; and the ability to dis￾criminate between differences in the GM crop and the comparator that result from the genetic modification and those differences in the plant’s germplasm, some of which may be attributed to soma-clonal variation introduced during tissue culture, and environmental or cultivation conditions. All such changes that might have health implications warrant further investigation, even if they can not only be attributed to the genetic modifi- cation, unless they are not manifested in subsequent generations foreseen for commercial cultivation. Any identified differences are then further assessed as to whether they might have adverse implications for human health in the range of exposure scenarios. The concept of substantial equivalence is thus the starting point and guiding concept for the safety assessment, not its conclusion (FAO/WHO, 2000; Codex Alimentarius Commission, 2003b). If there are no significant differ￾ences between the GM crop and the comparator or if there are differences that will, with reasonable certainty, not adversely affect health, the GM product is con￾sidered ‘as safe as’ its counterpart. This approach also applies to GM crops with more complex metabolic modifications, where no single parent crop might be a suitable comparator, but where single widely consumed substances, food constituents, ingredients, or other whole foods that are deemed safe under representative conditions of use may serve as comparators. Critics of the concept of substantial equivalence claim that current testing approaches do not sufficiently address putative unintended and unexpected effects and can not rule out the occurrence of potential long-term effects that result from sustained human exposure to such crops that might have subtle compositional chan￾ges that may be difficult to detect (Millstone et al., 1999). Furthermore, some critics maintain that there is a lack of detailed international standards guiding the choice of parameters to be measured in the comparative ana￾lysis and in the application of rigorous statistical analy￾sis, reducing the quality of individual assessments (SBC, 2001). Groups of international experts have reviewed the concept of substantial equivalence in the light of these criticisms. It was concluded that the concept still represents the best available assessment paradigm; no alternative approaches for the safety assessment of foods derived from GM crops have been proposed (FAO/WHO, 2000; Codex Alimentarius Commission, 2003b). Detailed international guidelines for choice of comparators and for best practices in statistical analysis have been and are being established under the auspices of the Organisation for Economic Coordination and Development (see also Section 4.1). In summary, the concept of substantial equivalence is widely accepted by international and national agencies as the best available guidance for the safety assessment of new GM crops. The approach recognises that foods are complex matrices containing tens of thousands of individual constituents, and that their safety assessment therefore requires a comparative approach focusing on those parameters deemed indicative of the normal functioning of the plant and its metabolism (including biosynthesis of any compound that might affect human health). As with all scientific concepts, the concept of substantial equivalence is evolving and, together with guidelines, making its application more systematic. The assessment helps to determine whether the GM crop is ‘as safe as’ its conventional counterpart. Dialogue between experts and civil society will contribute over time to further clarify and structure risk analysis strate￾gies to improve the salience of assessments to address concerns of policy makers and the public (Tait, 2001; Jasanoff, 2000; Schauzu, 2000). 3. Methods for toxicity testing Regulatory requirements for chemicals such as food additives and pesticides, many of which were first insti￾tuted in the 1970s, have led to the development of a battery of tests to assess the safety of chemicals in foods. Strategies for assessing the food safety of chemicals often combine three approaches: investigation of the structure/function relationship for indications of poten￾tial toxicity and allergenicity; in vitro assays with enzymes, receptor proteins, or cultured cell lines; and in vivo animal studies. The selection of animal studies is based on considerations including a molecule’s struc￾ture, function and in vitro toxicity results, as well as ethical criteria. Of these three distinct approaches, evi￾dence from animal tests is usually most indicative of potential toxic effects of a test substance in humans. However, the extrapolation of results from animal tests to humans is uncertain, unpredictable differences can include inter-species and inter-individual differences in metabolism, physiological processes, and lifestyle. These uncertainties are usually addressed through the use of uncertainty factors (see Section 2.2). Individual tox￾icological tests can be designed to be specific to test an hypothesis of a molecule’s toxic effect on one particular A. Ko¨nig et al. / Food and Chemical Toxicology 42 (2004) 1047–1088 1053

1054 A.Konig et al.Food and Chemical Toxicology 42 (2004)1047-1088 organ,a combination of specific endpoints,or they can for assessing the likely allergenicity of a novel protein be broad and non-targeted. (FAO/WHO,2001;Codex Alimentarius Commission, A combination of database screening,in vitro,and in 2003c;Kleter and Peijnenburg,2002).The merits and vivo testing approaches is therefore deployed in most limitations of this approach for assessing the safety of food safety testing strategies and provides assurance novel proteins introduced into GM crops are described that the tested food will be as safe as other foods that in more detail in Section 4.3.4. are routinely consumed.The characterisation of the test substance's physico-chemical properties and structure- 3.2.In vitro methods activity relationship that might be indicative of the pre- sence or absence of potential adverse health effects helps In vitro methods contribute to the safety assessment to frame and focus the testing approach.Several tests of chemicals,including food additives:they can in some established for chemicals are applicable to,or have been cases serve as indicators for specific toxic effects of dis- adapted for testing purified recombinant proteins or crete molecules and substances.There are several dif- other substances contained in foods derived from GM ferent types of in vitro methods;these include the in crops.Some animal methods have also been adapted to vitro simulation of digestive systems to assess stability; gain information pertaining to the safety of whole foods bioassays of the activity of purified enzymes;immorta- derived from GM crops.The following section will lised cultured cell lines:or in vitro reconstructions of provide a brief overview on the three distinct approa- receptor or membrane systems.The methods can serve ches developed for chemicals,highlighting which of either as screening systems to assess potential toxicity of these have been deployed in strategies to assess the a compound.or for investigations of a toxicological safety of foods derived from GM crops. mechanism underlying a specific effect observed in vivo or predicted from the structure of a molecule.Such 3.1.Investigating the relationship between structure and assays can serve for instance to assess whether mole- activity for indications of potential adverse effects cules bind to,inhibit,or stimulate proteins with specific functions.In vitro tests include tests that are claimed to The investigation of a substance's structure-activity be indicative of specific organ toxicity.An overview on relationship starts with a description of the defining the status of many in vitro methods in relation to the physico-chemical properties.Computers help to assess assessment of acute toxicity endpoints has been pub- whether a molecule shares characteristics of known lished (Walum.1998).A databank of in vitro techniques toxicants through screening databases with information in toxicology,called INVITTOX,has also been estab- on structural and physico-chemical properties of known lished (INVITTOX.2003).A critical discussion of the toxicants.Some toxicants show a clear structure-activity merits and limitations of in vitro methods is provided in relationship and their mechanism of toxicity is fully Eisenbrand et al.(2002). understood;other classes of toxicants just share com- Few in vitro tests are however validated formally,and mon structural elements or physico-chemical properties extrapolation of results from in vitro tests to in vivo that may be indicative of toxicity.Some physico- situations is often challenging,often their predictive chemical or structural characteristics of molecules are value has not been systematically assessed;uncertainties therefore indicative of a potential toxic effect.A have to be clearly stated.Advantages of the use of in molecule's physico-chemical properties help for instance vitro methods include that they are relatively cheap and the prediction of its propensity to intercalate in high-throughput.In vitro test results may be indicative DNA and interfere with its replication (Barratt,1998). of toxic effects;test systems relying on reconstituted Databases also exist listing proteins with (food) purified protein or cell components,or immortalised allergenic properties (see Table 1).Computer-based laboratory cultures of cell lines are,however,not methods have also been developed for the comparison representative of the functioning of such cell compo- of the primary amino acid sequences of proteins allow- nents or cells in living organisms.In vivo tests are ing identification of contiguous epitopes that might therefore required to confirm observations on toxicity mediate an allergenic effect.Limitations of the method from in vitro tests.The use of in vitro methods may include that non-continuous epitopes cannot be identi- become of greater importance with developments in the fied,and false positive matches through epitopes that area of genomic research and microarray systems to are similar but do not mediate allergenic effects.Any monitor changes in gene expression.Future develop- indication of toxicity or allergenicity obtained through ments in this area are discussed in more detail in Section computer screening has to be confirmed through other studies. The US Pharmacopeia describes simulated gastric and A computer-assisted search for similarity to proteins intestinal fluid preparations that have been used to known to cause adverse effects,such as protein toxins or assess the stability of proteins(US Pharmacopeia,1990; allergens,forms part of the current recommendations Astwood et al..1996).The test is used as one indicator

organ, a combination of specific endpoints, or they can be broad and non-targeted. A combination of database screening, in vitro, and in vivo testing approaches is therefore deployed in most food safety testing strategies and provides assurance that the tested food will be as safe as other foods that are routinely consumed. The characterisation of the test substance’s physico-chemical properties and structure￾activity relationship that might be indicative of the pre￾sence or absence of potential adverse health effects helps to frame and focus the testing approach. Several tests established for chemicals are applicable to, or have been adapted for testing purified recombinant proteins or other substances contained in foods derived from GM crops. Some animal methods have also been adapted to gain information pertaining to the safety of whole foods derived from GM crops. The following section will provide a brief overview on the three distinct approa￾ches developed for chemicals, highlighting which of these have been deployed in strategies to assess the safety of foods derived from GM crops. 3.1. Investigating the relationship between structure and activity for indications of potential adverse effects The investigation of a substance’s structure-activity relationship starts with a description of the defining physico-chemical properties. Computers help to assess whether a molecule shares characteristics of known toxicants through screening databases with information on structural and physico-chemical properties of known toxicants. Some toxicants show a clear structure–activity relationship and their mechanism of toxicity is fully understood; other classes of toxicants just share com￾mon structural elements or physico-chemical properties that may be indicative of toxicity. Some physico￾chemical or structural characteristics of molecules are therefore indicative of a potential toxic effect. A molecule’s physico-chemical properties help for instance the prediction of its propensity to intercalate in DNA and interfere with its replication (Barratt, 1998). Databases also exist listing proteins with (food) allergenic properties (see Table 1). Computer-based methods have also been developed for the comparison of the primary amino acid sequences of proteins allow￾ing identification of contiguous epitopes that might mediate an allergenic effect. Limitations of the method include that non-continuous epitopes cannot be identi- fied, and false positive matches through epitopes that are similar but do not mediate allergenic effects. Any indication of toxicity or allergenicity obtained through computer screening has to be confirmed through other studies. A computer-assisted search for similarity to proteins known to cause adverse effects, such as protein toxins or allergens, forms part of the current recommendations for assessing the likely allergenicity of a novel protein (FAO/WHO, 2001; Codex Alimentarius Commission, 2003c; Kleter and Peijnenburg, 2002). The merits and limitations of this approach for assessing the safety of novel proteins introduced into GM crops are described in more detail in Section 4.3.4. 3.2. In vitro methods In vitro methods contribute to the safety assessment of chemicals, including food additives; they can in some cases serve as indicators for specific toxic effects of dis￾crete molecules and substances. There are several dif￾ferent types of in vitro methods; these include the in vitro simulation of digestive systems to assess stability; bioassays of the activity of purified enzymes; immorta￾lised cultured cell lines; or in vitro reconstructions of receptor or membrane systems. The methods can serve either as screening systems to assess potential toxicity of a compound, or for investigations of a toxicological mechanism underlying a specific effect observed in vivo or predicted from the structure of a molecule. Such assays can serve for instance to assess whether mole￾cules bind to, inhibit, or stimulate proteins with specific functions. In vitro tests include tests that are claimed to be indicative of specific organ toxicity. An overview on the status of many in vitro methods in relation to the assessment of acute toxicity endpoints has been pub￾lished (Walum, 1998). A databank of in vitro techniques in toxicology, called INVITTOX, has also been estab￾lished (INVITTOX, 2003). A critical discussion of the merits and limitations of in vitro methods is provided in Eisenbrand et al. (2002). Few in vitro tests are however validated formally, and extrapolation of results from in vitro tests to in vivo situations is often challenging, often their predictive value has not been systematically assessed; uncertainties have to be clearly stated. Advantages of the use of in vitro methods include that they are relatively cheap and high-throughput. In vitro test results may be indicative of toxic effects; test systems relying on reconstituted purified protein or cell components, or immortalised laboratory cultures of cell lines are, however, not representative of the functioning of such cell compo￾nents or cells in living organisms. In vivo tests are therefore required to confirm observations on toxicity from in vitro tests. The use of in vitro methods may become of greater importance with developments in the area of genomic research and microarray systems to monitor changes in gene expression. Future develop￾ments in this area are discussed in more detail in Section 5. The US Pharmacopeia describes simulated gastric and intestinal fluid preparations that have been used to assess the stability of proteins (US Pharmacopeia, 1990; Astwood et al., 1996). The test is used as one indicator 1054 A. Ko¨nig et al. / Food and Chemical Toxicology 42 (2004) 1047–1088

A.Konig et al.Food and Chemical Toxicology 42 (2004)1047-1088 1055 as to whether the recombinant protein shares the whole foods to animals at doses that are large multiples characteristic of stability to digestion under these con- of the expected human exposure.Frequently,in feeding ditions that is common to many allergens (see Section trials with whole foods at the highest administered dose. 4.3.4) no adverse effect is observed.In animal studies where no adverse effect is observed.and where administered doses 3.3.Animal tests do not significantly exceed expected human exposure levels,it is not possible to account for uncertainties A variety of tests with laboratory animals have been regarding variations of susceptibility between species and developed for identifying and characterizing health between individuals within a population.Furthermore, hazards associated with exposure to single well-defined the food matrix in which the test material is adminis- chemicals and food additives.Tolerance studies are tered needs careful consideration:it may be difficult to sometimes used to confirm the absence of adverse effects ensure nutritional balance when diets contain high pro- at expected levels of exposure.Ethical considerations portions of novel foods or food extracts.Inadvertent should be an important driver in decisions on whether changes in nutritional status may result in adaptive chan- to conduct in vivo studies,in the choice of test species, ges that may mistakenly be attributed to adverse effects and in the design of the study protocol.Both tox- from intake from the GM crop and can be attributed to icological animal tests and tolerance studies are dis- changes resulting from the genetic modification. cussed in more detail below. Animal feeding studies with whole foods that take Animals can be used for the determination of acute account of these difficulties through careful design and toxicity of a substance,usually involving administration description of conditions under which the tests are car- of a large single dose followed by a short period of ried out and any remaining uncertainties have,however, observation.Sub-chronic toxicity,chronic toxicity,or been used to complement other tests for the safety carcinogenicity is tested in animals over prolonged per- assessments of foods derived from GM crops.Such tests iods of one or several months,or the lifetime of an ani- may provide useful information,in particular if doses mal.The use of these methods has recently been that are multiples of anticipated human exposure can be reviewed in detail by the European Concerted Action administered to animals (Hammond et al.,1996:Kuiper Food Safety in Europe (FOSIE)(Barlow et al.,2002). etal.,2001). They form the basis of toxicity testing of chemicals,and Studies in which animals (or,all be it rarely,human have provided valuable information for this purpose volunteers)are administered the expected level of intake (Kroes and Kozianowski,2002).In addition to the or low multiples of that level,so-called tolerance stud- standard tests listed in Appendix A,other studies could ies,can be used to complement other safety testing be carried out where deemed necessary to address approaches.Well-established protocols for tolerance immuno-toxicity;endocrine toxicity;individual organ studies of pharmaceuticals are available and can be toxicity;and toxico-kinetic investigations on absorp- adapted for this purpose.The highest dose used must be tion,distribution,metabolism,and excretion (ADME) without adverse nutritional effects and must respect the of a substance.Most of these methods have been stan- appropriate balance of nutrients required by the test dardised and OECD guidelines are available for their species.A classical range of parameters such as growth conduct and interpretation (see Appendix A;OECD, rate and feed efficiency are measured to identify possible 1993b). adverse effects on specific endpoints.The use of appro- These animal methods,if deemed necessary,can be priate controls is an important determinant of the adapted to test for potential adverse effects of recombi- validity of the results.For instance,if the novel com- nant proteins or novel compounds or metabolites ponent is normally present in a particular matrix,then introduced into crops through genetic modification (see that matrix would be an appropriate control.For the Section 4.3).It can however be difficult to obtain suffi- tolerance assessment of GM crops controls should cient quantities of purified recombinant proteins for include animal groups using the corresponding com- testing in animals over prolonged periods of time. parator.Similar methods can be used for animal and Adapting OECD guidelines and principles for testing human studies(see also Section 4.4.3). specific well defined chemicals to the assessment of whole foods,including whole foods derived from GM 3.4.Post-market monitoring crops,presents,however,several challenges.First,ani- mal tests for chemicals are usually designed to identify a Post-market monitoring systems have been estab- dose response relationship from which the consequences lished by several food companies for certain food pro- of exposure to low doses,typical of human exposure, ducts to act as early warning systems and to facilitate can be extrapolated.The highest administered dose is product recall in the event where health concerns might normally expected to produce some observable adverse be associated with a specific food.The organisation of effect.It is,however,often not possible to administer post-market monitoring is primarily the responsibility

as to whether the recombinant protein shares the characteristic of stability to digestion under these con￾ditions that is common to many allergens (see Section 4.3.4). 3.3. Animal tests A variety of tests with laboratory animals have been developed for identifying and characterizing health hazards associated with exposure to single well-defined chemicals and food additives. Tolerance studies are sometimes used to confirm the absence of adverse effects at expected levels of exposure. Ethical considerations should be an important driver in decisions on whether to conduct in vivo studies, in the choice of test species, and in the design of the study protocol. Both tox￾icological animal tests and tolerance studies are dis￾cussed in more detail below. Animals can be used for the determination of acute toxicity of a substance, usually involving administration of a large single dose followed by a short period of observation. Sub-chronic toxicity, chronic toxicity, or carcinogenicity is tested in animals over prolonged per￾iods of one or several months, or the lifetime of an ani￾mal. The use of these methods has recently been reviewed in detail by the European Concerted Action Food Safety in Europe (FOSIE) (Barlow et al., 2002). They form the basis of toxicity testing of chemicals, and have provided valuable information for this purpose (Kroes and Kozianowski, 2002). In addition to the standard tests listed in Appendix A, other studies could be carried out where deemed necessary to address immuno-toxicity; endocrine toxicity; individual organ toxicity; and toxico-kinetic investigations on absorp￾tion, distribution, metabolism, and excretion (ADME) of a substance. Most of these methods have been stan￾dardised and OECD guidelines are available for their conduct and interpretation (see Appendix A; OECD, 1993b). These animal methods, if deemed necessary, can be adapted to test for potential adverse effects of recombi￾nant proteins or novel compounds or metabolites introduced into crops through genetic modification (see Section 4.3). It can however be difficult to obtain suffi- cient quantities of purified recombinant proteins for testing in animals over prolonged periods of time. Adapting OECD guidelines and principles for testing specific well defined chemicals to the assessment of whole foods, including whole foods derived from GM crops, presents, however, several challenges. First, ani￾mal tests for chemicals are usually designed to identify a dose response relationship from which the consequences of exposure to low doses, typical of human exposure, can be extrapolated. The highest administered dose is normally expected to produce some observable adverse effect. It is, however, often not possible to administer whole foods to animals at doses that are large multiples of the expected human exposure. Frequently, in feeding trials with whole foods at the highest administered dose, no adverse effect is observed. In animal studies where no adverse effect is observed, and where administered doses do not significantly exceed expected human exposure levels, it is not possible to account for uncertainties regarding variations of susceptibility between species and between individuals within a population. Furthermore, the food matrix in which the test material is adminis￾tered needs careful consideration: it may be difficult to ensure nutritional balance when diets contain high pro￾portions of novel foods or food extracts. Inadvertent changes in nutritional status may result in adaptive chan￾ges that may mistakenly be attributed to adverse effects from intake from the GM crop and can be attributed to changes resulting from the genetic modification. Animal feeding studies with whole foods that take account of these difficulties through careful design and description of conditions under which the tests are car￾ried out and any remaining uncertainties have, however, been used to complement other tests for the safety assessments of foods derived from GM crops. Such tests may provide useful information, in particular if doses that are multiples of anticipated human exposure can be administered to animals (Hammond et al., 1996; Kuiper et al., 2001). Studies in which animals (or, all be it rarely, human volunteers) are administered the expected level of intake or low multiples of that level, so-called tolerance stud￾ies, can be used to complement other safety testing approaches. Well-established protocols for tolerance studies of pharmaceuticals are available and can be adapted for this purpose. The highest dose used must be without adverse nutritional effects and must respect the appropriate balance of nutrients required by the test species. A classical range of parameters such as growth rate and feed efficiency are measured to identify possible adverse effects on specific endpoints. The use of appro￾priate controls is an important determinant of the validity of the results. For instance, if the novel com￾ponent is normally present in a particular matrix, then that matrix would be an appropriate control. For the tolerance assessment of GM crops controls should include animal groups using the corresponding com￾parator. Similar methods can be used for animal and human studies (see also Section 4.4.3). 3.4. Post-market monitoring Post-market monitoring systems have been estab￾lished by several food companies for certain food pro￾ducts to act as early warning systems and to facilitate product recall in the event where health concerns might be associated with a specific food. The organisation of post-market monitoring is primarily the responsibility A. Ko¨nig et al. / Food and Chemical Toxicology 42 (2004) 1047–1088 1055

1056 A.Konig et al.Food and Chemical Toxicology 42 (2004)1047-1088 of the manufacturer of the food.Methods vary from and to link such variations to health outcomes (FSA, establishing channels of communication in the firm to 2002).Possible requirements for post-market monitor- receive direct consumer feedback on the product,to the ing of GM crops with more complex modifications, repurchase of products to determine the quality of the including altered nutritional value,are discussed in product on the supermarket shelf. Section 4.6. Post-market monitoring programmes may serve to confirm the absence of specific adverse health effects of certain products after they have been marketed.The 4.The safety assessment of foods derived from GM feasibility and validity of post-market monitoring crops depends on the health endpoint of interest and on the way the product is marketed.First,post-market mon- Safety considerations for foods derived from GM itoring of manifestation of adverse effects from intake of crops are fundamentally the same as those for conven- specific foods is most feasible if it is driven by an tional foods or other types of novel foods (Cockburn, hypothesis on a potential and specific adverse effect that 2002).Since the genetic improvement of crops has is acute and has distinctive symptoms.Acute adverse always been the aim of plant selection and breeding, effects generally associated with relatively high intakes 'traditional'approaches are appropriate in order to of a substance,or allergic reactions,are likely to become assess the safety of foods derived from all GM crops, apparent by post-market monitoring for spontaneous regardless of the crop species or the trait introduced by events.However,long-term or rare effects require genetic modification. generally a more targeted and intrusive study design. This section sets out a systematic stepwise approach Randomised controlled human trials could be used to on how to select appropriate combinations of test investigate possible medium/long term effects,but the methods to assess the safety of foods derived from GM wide variation in diets and dietary components from crops(Figs.I and 2).The objective of the assessment is day to day and year to year should be recognised.While to determine whether these new foods are at least as safe clinical studies in humans may provide wider assurance as foods produced from conventional crops;this of safety of whole foods.they cannot fully reproduce approach hence offers a high level of safety assurance. the diversity of the populations who will consume the The outlined approach serves to structure case-by- marketed product.The possibility therefore remains case assessments of specific products;it provides gui- that unpredicted side effects may occur in some sectors dance on how to design a test programme for the safety of the population,such as those with certain disease assessment of a GM crop that is tailored to the specific conditions or with particular genetic characteristics.In characteristics of the parent crop and the introduced addition,risk assessment relies on an estimate of expo- trait(s).The safety assessment focuses on the new gene sure to the food,which is variable and subject to product(s)and of whole foods derived from the GM uncertainty before the food is marketed.Critical issues crop.Both intended and potential unintended effects to the success of a post-market monitoring programme from the genetic modification are taken into account. relating to the marketing approach of the foods are the The assessment involves the following steps:(i)char- ability to estimate exposure with reasonable accuracy acterisation of the parent crop;(ii)characterisation of (traceability)and to match any reported effects to the the donor organism(s)from which any recombinant consumption of the material (Wal et al.,2003).For DNA sequences are derived,the transformation pro- identity-preserved products this may be feasible, cess,and the introduced recombinant DNA sequences; whereas for commodities it is much more difficult,if not (iii)safety assessment of the introduced gene products impossible. (proteins and metabolites);and (iv)food safety assess- To date,no GM crop has been placed on the market ment of whole food derived from,or edible part of,the for which post-market monitoring was deemed neces- GM crop. sary.The current safety testing strategy has been con- The assessment focuses on any changes introduced sidered sufficiently predictive for these approvals. through the genetic modification,including introduced Problems limiting the interpretation of post-market genes and gene products,and potentially altered levels monitoring of foods derived from commodity crops of endogenous compounds or the formation of new have been highlighted(FAO/WHO,2000).The United metabolites.Methods for the detection of unexpected Kingdom Food Standards Agency has commissioned a changes in the composition due to the genetic modifi- study to examine the feasibility of using supermarket cation process are discussed and evaluated in the paper and household survey data for post-market monitoring by Cellini et al.(2004).Possible consequences of trans- of novel foods.This study assesses the government's fer of the recombinant sequences to gastrointestinal ability to detect variations of food purchasing and microflora or to humans should also be assessed and are consumption at the district level in Great Britain,as evaluated in the paper by Van den Eede et al.(2004). this is seen as an indicator for the feasibility to detect The assembled information may also help to identify

of the manufacturer of the food. Methods vary from establishing channels of communication in the firm to receive direct consumer feedback on the product, to the repurchase of products to determine the quality of the product on the supermarket shelf. Post-market monitoring programmes may serve to confirm the absence of specific adverse health effects of certain products after they have been marketed. The feasibility and validity of post-market monitoring depends on the health endpoint of interest and on the way the product is marketed. First, post-market mon￾itoring of manifestation of adverse effects from intake of specific foods is most feasible if it is driven by an hypothesis on a potential and specific adverse effect that is acute and has distinctive symptoms. Acute adverse effects generally associated with relatively high intakes of a substance, or allergic reactions, are likely to become apparent by post-market monitoring for spontaneous events. However, long-term or rare effects require generally a more targeted and intrusive study design. Randomised controlled human trials could be used to investigate possible medium/long term effects, but the wide variation in diets and dietary components from day to day and year to year should be recognised. While clinical studies in humans may provide wider assurance of safety of whole foods, they cannot fully reproduce the diversity of the populations who will consume the marketed product. The possibility therefore remains that unpredicted side effects may occur in some sectors of the population, such as those with certain disease conditions or with particular genetic characteristics. In addition, risk assessment relies on an estimate of expo￾sure to the food, which is variable and subject to uncertainty before the food is marketed. Critical issues to the success of a post-market monitoring programme relating to the marketing approach of the foods are the ability to estimate exposure with reasonable accuracy (traceability) and to match any reported effects to the consumption of the material (Wal et al., 2003). For identity-preserved products this may be feasible, whereas for commodities it is much more difficult, if not impossible. To date, no GM crop has been placed on the market for which post-market monitoring was deemed neces￾sary. The current safety testing strategy has been con￾sidered sufficiently predictive for these approvals. Problems limiting the interpretation of post-market monitoring of foods derived from commodity crops have been highlighted (FAO/WHO, 2000). The United Kingdom Food Standards Agency has commissioned a study to examine the feasibility of using supermarket and household survey data for post-market monitoring of novel foods. This study assesses the government’s ability to detect variations of food purchasing and consumption at the district level in Great Britain, as this is seen as an indicator for the feasibility to detect and to link such variations to health outcomes (FSA, 2002). Possible requirements for post-market monitor￾ing of GM crops with more complex modifications, including altered nutritional value, are discussed in Section 4.6. 4. The safety assessment of foods derived from GM crops Safety considerations for foods derived from GM crops are fundamentally the same as those for conven￾tional foods or other types of novel foods (Cockburn, 2002). Since the genetic improvement of crops has always been the aim of plant selection and breeding, ‘traditional’ approaches are appropriate in order to assess the safety of foods derived from all GM crops, regardless of the crop species or the trait introduced by genetic modification. This section sets out a systematic stepwise approach on how to select appropriate combinations of test methods to assess the safety of foods derived from GM crops (Figs. 1 and 2). The objective of the assessment is to determine whether these new foods are at least as safe as foods produced from conventional crops; this approach hence offers a high level of safety assurance. The outlined approach serves to structure case-by￾case assessments of specific products; it provides gui￾dance on how to design a test programme for the safety assessment of a GM crop that is tailored to the specific characteristics of the parent crop and the introduced trait(s). The safety assessment focuses on the new gene product(s) and of whole foods derived from the GM crop. Both intended and potential unintended effects from the genetic modification are taken into account. The assessment involves the following steps: (i) char￾acterisation of the parent crop; (ii) characterisation of the donor organism(s) from which any recombinant DNA sequences are derived, the transformation pro￾cess, and the introduced recombinant DNA sequences; (iii) safety assessment of the introduced gene products (proteins and metabolites); and (iv) food safety assess￾ment of whole food derived from, or edible part of, the GM crop. The assessment focuses on any changes introduced through the genetic modification, including introduced genes and gene products, and potentially altered levels of endogenous compounds or the formation of new metabolites. Methods for the detection of unexpected changes in the composition due to the genetic modifi- cation process are discussed and evaluated in the paper by Cellini et al. (2004). Possible consequences of trans￾fer of the recombinant sequences to gastrointestinal microflora or to humans should also be assessed and are evaluated in the paper by Van den Eede et al. (2004). The assembled information may also help to identify 1056 A. Ko¨nig et al. / Food and Chemical Toxicology 42 (2004) 1047–1088